Journal: PLoS ONE

Article Title: Modeling Initiation of Ewing Sarcoma in Human Neural Crest Cells

doi: 10.1371/journal.pone.0019305

Figure Lengend Snippet: Core target genes significantly regulated by EWS-FLI1 in BM-MSC * , ESFT cell lines ** , and hNC-MSC.

Article Snippet: Western blots were performed using standard procedures and the following antibodies at 1∶1000 dilutions: V5 (Invitrogen), BMI-1 (Millipore); EZH2, GAPDH, Cyclin D1 (Cell Signaling Technology); p16, Actin, (Santa Cruz Biotechnology); p21 (Abcam); p53 (1∶2000, Cell signaling).

Techniques:

Journal: PLoS ONE

Article Title: Modeling Initiation of Ewing Sarcoma in Human Neural Crest Cells

doi: 10.1371/journal.pone.0019305

Figure Lengend Snippet: ( A ) EWS-FLI1 (EF-NC) and control vector (CV-NC) transduced hNC-MSC were passaged in serum-containing media for 3 weeks and morphology imaged by immunocytochemistry. EF-NC cells were smaller and less mesenchymal in appearance than CV-NC cells (scale bar = 100 µm). ( B ) RT-PCR confirms down regulation of NCSC genes B3GAT1 (HNK-1) and NGFR (p75) in CV-NC cells after 2 weeks. In contrast, NCSC and EWS-FLI1 target gene expression remained high in EF-NC cells (results for 3 independent experiments are shown). ( C ) Transduced, EGFP+ cells were isolated 6 weeks after transduction and analyzed by western blot. Data from 3 independent experiments show consistent up regulation of BMI-1 and EZH2 and repression of p16 in EF-NC cells. ( D ) After 6 weeks BMI-1, EZH2 and p16 expression in EF-NC cells were equivalent to freshly isolated hNCSC. In contrast, CV-NC cells down regulated polycomb proteins and up regulated p16.

Article Snippet: Western blots were performed using standard procedures and the following antibodies at 1∶1000 dilutions: V5 (Invitrogen), BMI-1 (Millipore); EZH2, GAPDH, Cyclin D1 (Cell Signaling Technology); p16, Actin, (Santa Cruz Biotechnology); p21 (Abcam); p53 (1∶2000, Cell signaling).

Techniques: Control, Plasmid Preparation, Immunocytochemistry, Reverse Transcription Polymerase Chain Reaction, Targeted Gene Expression, Isolation, Transduction, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Modeling Initiation of Ewing Sarcoma in Human Neural Crest Cells

doi: 10.1371/journal.pone.0019305

Figure Lengend Snippet: ( A ) Inducible BMI-1 knockdown lentiviral vector. ( B ) Red fluorescent protein expression following doxycyline treatment confirmed induction of inert non-silencing (NS) and BMI-1-targeted (BMI1 kd) shRNA in transduced cells (scale bar = 100 µm). ( C ) Western blot analysis confirmed BMI-1 knockdown in BMI1 kd cells accompanied by de-repression of p16 and concomitant down regulation of EZH2. ( D ) BMI-1 knockdown cells also expressed reduced levels of Cyclin D1 compared to control (NS) cells. ( E ) Senescence-associated beta-galactosidase staining 1 week post-doxycyline induction shows that BMI-1 kd cells were senescent (scale bar = 100 µm).

Article Snippet: Western blots were performed using standard procedures and the following antibodies at 1∶1000 dilutions: V5 (Invitrogen), BMI-1 (Millipore); EZH2, GAPDH, Cyclin D1 (Cell Signaling Technology); p16, Actin, (Santa Cruz Biotechnology); p21 (Abcam); p53 (1∶2000, Cell signaling).

Techniques: Knockdown, Plasmid Preparation, Expressing, shRNA, Western Blot, Control, Staining

Journal: Cellular and Molecular Bioengineering

Article Title: A Stable Pep2-proapoptotic Peptide Inducing Apoptosis of Acute Myeloid Leukemia Cells by Down-Regulating EZH2

doi: 10.1007/s12195-019-00605-z

Figure Lengend Snippet: Effect of 2PP7-Pep2-KLAK VLPs on EZH2 in THP-1 cells. THP-1 cells were exposed to 80 nM of 2PP7-Pep2 VLPs or 2PP7-Pep2-KLAK VLPs. After 48 h, the cells were collected by centrifugation. The mRNA and protein levels of EZH2 were detected by RT-qPCR (a) and WB (b), respectively. Also, the expression of H3K27me3 was quantified by WB (b).

Article Snippet: Proteins (40 mg) were fractioned by SDS-PAGE and the target proteins were analyzed using the anti-EZH2 (ab186006), GAPDH (ab9485, Abcam), H3 (D1H2) or H3K27me3 (C36B11, Cell signaling) antibodies.

Techniques: Centrifugation, Quantitative RT-PCR, Expressing

Journal: International immunopharmacology

Article Title: Characterization of PANoptosis-related genes with immunoregulatory features in osteoarthritis.

doi: 10.1016/j.intimp.2024.112889

Figure Lengend Snippet: Fig. 5. The correlation between key Differentially Expressed Genes Associated with Panoptosis (DE-PRGs) and immune cells. (A–G) The association of CDKN1A, EZH2, MEG3, NR4A1, PIK3R2, S100A8, and SYVN1 with immune cells.

Article Snippet: After blocking with 5 % fat-free dry milk for 60 min at room temperature, the membranes were then exposed overnight at 4 ◦C to primary antibodies targeting CDKN1A (Proteintech, China), EZH2 (Proteintech, China), NR4A1 (Proteintech, China), PIK3R2 (Proteintech, China), MEG3 (Proteintech, China), S100A8 (Proteintech, China), SYVN1 (Proteintech, China), and β-actin (1:5000).

Techniques:

Journal: International immunopharmacology

Article Title: Characterization of PANoptosis-related genes with immunoregulatory features in osteoarthritis.

doi: 10.1016/j.intimp.2024.112889

Figure Lengend Snippet: Fig. 6. Potential drug screening and primary structure of proteins. (A) CMap analysis revealed the mechanism of action associated with small-molecule compounds; (B–H) Primary protein structures of CDKN1A, EZH2, MEG3, NR4A1, PIK3R2, S100A8, and SYVN1; (I) Screening of the 3D structure of APHA-compound-8 through the PubChem open chemical database.

Article Snippet: After blocking with 5 % fat-free dry milk for 60 min at room temperature, the membranes were then exposed overnight at 4 ◦C to primary antibodies targeting CDKN1A (Proteintech, China), EZH2 (Proteintech, China), NR4A1 (Proteintech, China), PIK3R2 (Proteintech, China), MEG3 (Proteintech, China), S100A8 (Proteintech, China), SYVN1 (Proteintech, China), and β-actin (1:5000).

Techniques: Drug discovery

Journal: International immunopharmacology

Article Title: Characterization of PANoptosis-related genes with immunoregulatory features in osteoarthritis.

doi: 10.1016/j.intimp.2024.112889

Figure Lengend Snippet: Fig. 7. Molecular docking models. (A-G) Molecular docking models of CDKN1A, EZH2, MEG3, NR4A1, PIK3R2, S100A8, and SYVN1 with the potential drug (APHA- compound-8).

Article Snippet: After blocking with 5 % fat-free dry milk for 60 min at room temperature, the membranes were then exposed overnight at 4 ◦C to primary antibodies targeting CDKN1A (Proteintech, China), EZH2 (Proteintech, China), NR4A1 (Proteintech, China), PIK3R2 (Proteintech, China), MEG3 (Proteintech, China), S100A8 (Proteintech, China), SYVN1 (Proteintech, China), and β-actin (1:5000).

Techniques:

Journal: International immunopharmacology

Article Title: Characterization of PANoptosis-related genes with immunoregulatory features in osteoarthritis.

doi: 10.1016/j.intimp.2024.112889

Figure Lengend Snippet: Fig. 8. Validation by Western blot experiments. (A-H) Comparative expression analysis of CDKN1A, EZH2, MEG3, NR4A1, PIK3R2, S100A8, and SYVN1 between the normal and OA groups, all showing statistically significant differences. (***P ≤0.001).

Article Snippet: After blocking with 5 % fat-free dry milk for 60 min at room temperature, the membranes were then exposed overnight at 4 ◦C to primary antibodies targeting CDKN1A (Proteintech, China), EZH2 (Proteintech, China), NR4A1 (Proteintech, China), PIK3R2 (Proteintech, China), MEG3 (Proteintech, China), S100A8 (Proteintech, China), SYVN1 (Proteintech, China), and β-actin (1:5000).

Techniques: Biomarker Discovery, Western Blot, Expressing

Journal: Disease Models & Mechanisms

Article Title: Altered huntingtin−chromatin interactions predict transcriptional and epigenetic changes in Huntington's disease

doi: 10.1242/dmm.052282

Figure Lengend Snippet: ChIP-seq analysis reveals early changes in H3K27me3 near developmentally important genes in Htt Q111/+ mice. (A) Volcano plots summarizing differential histone modifications for H3K27me3, H3K9me3 and H3K4me3, and differential occupancy for EZH2 in striatal tissue from 4-month-old in Htt Q111/+ versus Htt +/+ mice. The x -axis indicates the log 2 FC and the y -axis indicates the -log 10 ( P -value) for the comparison of peak-read depth between genotypes. Regions with greater levels or occupancy in Htt +/+ mice are shown in blue, regions with greater levels or occupancy in Htt Q111/+ mice are shown in pink. Dashed horizontal lines indicate an FDR of 0.1; vertical lines are Log 2 FC=±0.26. (B) Network depiction of Gene Ontology Biological Processes enrichment near differentially methylated regions with reduced H3K27me3 in Htt Q111/+ mice. (C) Gene Ontology Molecular Function enrichment near differentially methylated regions with reduced H3K27me3 in Htt Q111/+ mice. (D) Enrichment of 495 genes in nominal differentially methylated regions with reduced H3K27me3 in Htt Q111/+ mice (unadjusted P <0.005) among consensus transcription factor (TF)/target gene databases, i.e. ENCODE and ChEA Consensus TFs from the Enrichr package. Robustly enriched transcription factor lists include: SUZ12 CHEA (93/1684; P adj =8.3e13; EZH2 CHEA (19/237; p adj =2.0e04); SMAD4 CHEA (27/584; P adj =2.6E02); REST CHEA (48/1280; P adj =2.8e02). (E) Enrichment of EZH2 and indicated chromatin mark ChIP-seq peaks (MACS FDR<0.05) in HTT ChIP-seq peak regions. The y -axis indicates the log-transformed fold change (enrichment or depletion) in the number of overlapping base pairs compared to the average of 100,000 re-sampling permutations of genomic coordinates. Plotting density indicates the P -value, derived from these same permutations.

Article Snippet: DNA was sonicated to an average fragment length of 300−500 bp, and 25 μg chromatin plus 200 ng Drosophila spike-in chromatin was incubated with 4 μg antibody targeting EZH2, H3K27ac, H3K27me3 or H3K4me3 (Active Motif catalog numbers 39901, 39133, 39155 and 399159, respectively).

Techniques: ChIP-sequencing, Comparison, Methylation, Transformation Assay, Sampling, Derivative Assay

Journal: Disease Models & Mechanisms

Article Title: Altered huntingtin−chromatin interactions predict transcriptional and epigenetic changes in Huntington's disease

doi: 10.1242/dmm.052282

Figure Lengend Snippet: Increased HTT occupancy is negatively associated with H3K27me3 and positively associated with H3K4me3 near genes that are dysregulated in HD. (A-C) Each plot indicates the fold change of HTT occupancy ( x -axis; Htt Q111/Q111 vs Htt +/+ ) compared to the fold change in the same regions for H3K27me3 (A), H3K4me3 (B) and EZH2 (C) on the y -axis ( Htt Q111/+ vs Htt +/+ ). Analysis is restricted to 263 HTT peaks located ±20 kb from the TSSs of genes that are significantly up- or downregulated in the striatum of 6-month-old knock-in mouse models of HD mutations ( Htt Q111/+ vs Htt Q20/+ ). Each circle represents a single HTT ChIP-seq peak. Purple and yellow colored circles indicate the fold change in gene expression in the striatum of heterozygous HD knock-in mice, with upregulated genes in yellow and downregulated genes in purple (see color map). Circle sizes correspond to P -values for differential expression.

Article Snippet: DNA was sonicated to an average fragment length of 300−500 bp, and 25 μg chromatin plus 200 ng Drosophila spike-in chromatin was incubated with 4 μg antibody targeting EZH2, H3K27ac, H3K27me3 or H3K4me3 (Active Motif catalog numbers 39901, 39133, 39155 and 399159, respectively).

Techniques: Knock-In, ChIP-sequencing, Gene Expression, Quantitative Proteomics

Journal: bioRxiv

Article Title: Altered Huntingtin-Chromatin Interactions Predict Transcriptional and Epigenetic Changes in Huntington’s Disease

doi: 10.1101/2020.06.04.132571

Figure Lengend Snippet: A: Volcano plots summarizing differentially histone modifications for H3K27me3 and H3K4me3 and differential occupancy for EZH2 in striatal tissue from four-month-old in Htt Q111/+ vs. Htt +/+ mice. The x-axis indicates the log 2 FC and the y-axis indicates the −log 10 (p-value). Regions with greater levels or occupancy in the Htt +/+ mice are shown in orange, regions with lower levels/occupancy are indicated in blue. The dashed horizontal line indicates an FDR of 0.05, and the solid line represents a nominal p-value of 0.05. B: Enrichment of EZH2, H3K27me3 and H3K4me3 ChIP-seq peaks (MACS FDR < 0.05) at WT-specific, Q111-specific, and Shared HTT ChIP-seq peaks. Y-axis indicates the log-transformed fold change (enrichment or depletion) in the number of overlapping base pairs compared to the average from 100,000 re-sampling permutations of genomic coordinates. Plotting color indicates the p-value, derived from these same permutations. C: Gene Ontology Biological Processes enriched near differentially methylated regions with reduced H3K27me3 in Htt Q111/+ mice (GREAT ). X-axis, p-value from binomial test over genomic regions; plot color, p-value from the hypergeometric test over genes results; plot size, hypergeometric test fold-enrichment.

Article Snippet: DNA was sonicated to an average fragment length of 300-500bp, and 25ug chromatin plus 200ng Drosophila spike-in chromatin was incubated with antibody targeting EZH2, H3K27me3 or H3K4me3 (Active Motif catalog numbers 39901, 39155 and 399159, respectively).

Techniques: ChIP-sequencing, Transformation Assay, Sampling, Derivative Assay, Methylation

Journal: bioRxiv

Article Title: Altered Huntingtin-Chromatin Interactions Predict Transcriptional and Epigenetic Changes in Huntington’s Disease

doi: 10.1101/2020.06.04.132571

Figure Lengend Snippet: Each plot indicates the fold change of HTT occupancy in Htt Q111/+ / Htt +/+ mice (x-axis) compared to the fold change in the same regions for H3K27me3 (A), EZH2 (B), and H3K4me3 (C) (y-axis). Analysis is restricted to 263 HTT peaks located +/- 20 kb from the TSSs of genes that are significantly up- or down-regulated in the striatum of six-month-old knock-in mouse models of HD mutations. Each point represents a single HTT ChIP-seq peak and is labeled with the name of the adjacent differentially expressed gene. Point color indicates the fold change in gene expression in the striatum of HD knock-in mice (up = yellow; down = purple), and point size corresponds to the p-value for differential expression.

Article Snippet: DNA was sonicated to an average fragment length of 300-500bp, and 25ug chromatin plus 200ng Drosophila spike-in chromatin was incubated with antibody targeting EZH2, H3K27me3 or H3K4me3 (Active Motif catalog numbers 39901, 39155 and 399159, respectively).

Techniques: Knock-In, ChIP-sequencing, Labeling, Gene Expression, Quantitative Proteomics